Syzygium aromaticum essential oil supplementation during in vitro bovine oocyte maturation improves parthenogenetic embryonic development

Santos, Maria Valéria de Oliveira; Nascimento, Lucas Emanuel; Praxedes, Érika Almeida; Borges, Alana Azevedo; Silva, Alexandre Rodrigues; Bertini, Luciana Medeiros; Pereira, Alexsandra Fernandes

doi:10.1016/j.theriogenology.2019.01.031

Cited by 26 publications

(12 citation statements)

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“…Moreover, for evaluation of an oxidative stress by quantification of the intracellular ROS levels, cells were stained with the fluorescent probe H 2 DCFDA, according to a method described by Santos et al (2019). Thawed cells were washed with PBS and placed into polystyrene culture dishes treated for cell adhesion containing 500 µL of 5 µM H 2 DCFDA.…”

Section: Influence Of Cryopreservation On the Quality Of Fibroblast Lmentioning

confidence: 99%

“…Finally, for the assessment of ΔΨm, cells were stained using 500 nM of the fluorescent probe MitoTracker Red Ò (CMXRos), according to a method described by Santos et al (2019). The procedure, incubation, and evaluation of the ten images (two/animal) were performed as described for the quantification of ROS.…”

Section: Influence Of Cryopreservation On the Quality Of Fibroblast Lmentioning

confidence: 99%

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Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Borges

Lira

Nascimento

et al. 2020

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Background Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. Methods Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 2% antibiotic–antimycotic solution under a controlled atmosphere (38.5 °C, 5% CO2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Results All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts which presented oval nuclei, a fusiform shape and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While there was no difference observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2 ± 12.1%) when compared to that in the first and third passage (100.0 ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). Conclusions This study is the first report on isolation, characterization and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.

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Section: Influence Of Cryopreservation On the Quality Of Fibroblast Lmentioning

confidence: 99%

Section: Influence Of Cryopreservation On the Quality Of Fibroblast Lmentioning

confidence: 99%

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Borges

Lira

Nascimento

et al. 2020

PeerJ

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“…Moreover, our group recently demonstrated that the essential oil of S. aromaticum (EOSA) had a beneficial effect on the in vitro maturation of bovine oocytes (Santos et al, ); hence, we hypothesised that similar effects could be observed for bovine spermatozoa. The objective of the present study was to assess the protective effects of the EOSA on bovine epididymal spermatozoa quality parameters and ROS levels.…”

Section: Introductionmentioning

confidence: 86%

“…The EOSA was extracted by hydrodistillation of the flower buds using a Clevenger‐type apparatus (Craveiro, Matos, & Alencar, ). Briefly, this system consisted of a Clevenger adapter attached to a round‐bottom flask containing approximately 118.8 g of clove buds and distilled water warmed to 100°C (Santos et al, ). After 4 hr of extraction, the EOSA was dried by percolation through anhydrous Na 2 SO 4 .…”

Section: Methodsmentioning

confidence: 99%

Antioxidant effects of the essential oil ofSyzygium aromaticumon bovine epididymal spermatozoa

et al. 2019

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Focusing on its application in reproductive biotechnology, we evaluated the effects of the essential oil of Syzygium aromaticum (EOSA) on bovine epididymal sperm quality variables, including morphology, membrane functional integrity, membrane structural integrity, mitochondrial activity, metabolic activity, motility and oxidative stress by reactive oxygen species (ROS) levels. Bovine spermatozoa from eight males were incubated into the following groups: EOSA0 (without EOSA), EOSA10 (10 μg/ml of EOSA), EOSA15 (15 μg/ml of EOSA) and EOSA20 (20 μg/ml of EOSA); the incubation time with and without the EOSA was 1 or 6 hr. None of the sperm quality variables presented difference among the EOSA concentrations. However, the incubation time had a significant effect on the membrane functional integrity, membrane structural integrity, mitochondrial activity, progressive motility and some kinetic parameters. The effect of interaction among EOSA and incubation time was significant only on ROS levels. Spermatozoa incubated in the presence of 15 μg/ml of the EOSA for 1 hr had significantly reduced ROS levels compared with all other groups in the same time. In conclusion, the EOSA at a concentration of 15 µg/ml has antioxidant effects and protects bovine epididymal spermatozoa; hence, the EOSA may potentially be used in the field of reproductive biotechnology.

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“…Manuscript to be reviewed Santos et al (2019). Thawed cells were washed with PBS and placed into polystyrene culture dishes treated for cell adhesion containing 500 µL of µM H DCFDA.…”

Section: Influence Of Cryopreservation On the Quality Of Fibroblast Lmentioning

confidence: 99%

Peer Review #2 of "Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines (v0.1)"

Selokar¹

2020

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Background. Biobanking of cell lines is a promising tool of support for wildlife conservation. In particular, the ability to preserve fibroblast cell lines derived from collared peccaries is of significance as these wild mammals are unique to the Americas and play a large role in maintaining the ecosystem. We identified collared peccary fibroblasts by immunofluorescence and evaluated their morphology, growth, and adherence capacity. Further, we monitored the viability and metabolic activity of the fibroblasts to determine the effects of passage number and cryopreservation on establishment of cell lines. Methods. Skin biopsies were collected from the peripheral ear region from five adult animals in captivity. Initially, cells were isolated from fragments and cultured in the Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and 2% antibiotic-antimycotic solution under a controlled atmosphere (38.5°C, 5% CO 2). We evaluated the maintenance of primary cells for morphology, adherence capacity of explants, explants in subconfluence, cell growth, and absence of contamination. Moreover, we identified the fibroblast cells by immunofluorescence. Additionally, to evaluate the influence of the number of passages (first, third, and tenth passage) and cryopreservation on establishment of cell lines, fibroblasts were analysed for the viability, metabolic activity, population doubling time (PDT), levels of reactive oxygen species (ROS), and mitochondrial membrane potential (ΔΨm). Results. All explants (20/20) adhered to the dish in 2.4 days ± 0.5 with growth around the explants in 4.6 days ± 0.7, and subconfluence was observed within 7.8 days ± 1.0. Moreover, by morphology and immunocytochemistry analyses, cells were identified as fibroblasts presenting oval nuclei, a fusiform shape, and positive vimentin staining. No contamination was observed after culture without antibiotics and antifungals for 30 days. While no difference was observed for cell viability after the passages (first vs. third: P = 0.98; first vs. tenth: P = 0.76; third vs. tenth: P = 0.85), metabolic activity was found to be reduced in the tenth passage (23.2% ± 12.1%), when compared to that in the first and third passage (100.0% ± 24.4%, P = 0.006). Moreover, the cryopreservation did not influence the viability (P = 0.11), metabolic activity (P = 0.77), or PDT (P = 0.11). Nevertheless, a greater ΔΨm (P = 0.0001) was observed for the cryopreserved cells (2.12 ± 0.14) when compared to that in the non-cryopreserved cells (1.00 ± 0.05). Additionally, the cryopreserved cells showed greater levels of intracellular ROS after thawing (1.69 ± 0.38 vs. 1.00 ± 0.22, P = 0.04). Conclusions. This study is the first report on isolation, characterization, and cryopreservation of fibroblasts from collared peccaries. We showed that adherent cultures were efficient for obtaining fibroblasts, which can be used as donor cells for nuclei for species cloning and other applications.

show abstract

Syzygium aromaticum essential oil supplementation during in vitro bovine oocyte maturation improves parthenogenetic embryonic development

Cited by 26 publications

References 38 publications

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines

Antioxidant effects of the essential oil ofSyzygium aromaticumon bovine epididymal spermatozoa

Peer Review #2 of "Isolation, characterization, and cryopreservation of collared peccary skin-derived fibroblast cell lines (v0.1)"

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